GLP-2-carrying exosomes alleviate osteoporosis by upregulating miR-378a-3p to inhibit osteoclastic differentiation and NF-κB-MAPK pathway.

BACKGROUND: Osteoporosis, characterized by excessive osteoclast activity, remains a major health challenge. This study investigated the therapeutic potential of exosomes derived from glucagon-like peptide-2 (GLP-2)-overexpressing macrophages in osteoporosis, with specific focus on miR-378a-3p-mediated regulation of osteoclastogenesis and inflammatory signaling pathways. METHODS: GLP-2 was overexpressed in mouse RAW264.7 cells. Exosomes were extracted and characterized. In vitro, effects on osteoclast differentiation were assessed using Cell Counting Kit-8, tartrate-resistant acid phosphatase staining, and quantitative real-time polymerase chain reaction. Differentially expressed miRNAs were screened via bioinformatics analysis. In vivo, an osteoporosis rat model was induced by bilateral ovariectomy. Histopathological evaluation of femoral tissue was performed via Hematoxylin-Eosin and tartrate resistant acid phosphatase staining. Bone microarchitecture, inflammatory cytokines, and signaling pathways were evaluated using micro-CT, enzyme-linked immunosorbent assay, and western blotting. Moreover, miR-378a-3p antagonist was applied to investigate the role of miR-378a-3p in osteoporosis. RESULTS: GLP-2-carrying exosomes inhibited osteoclast differentiation in vitro and improved bone microarchitecture in vivo. Bioinformatics analysis identified miR-378a-3p as a hub miRNA and miR-378a-3p was downregulated in osteoporosis. In vivo, GLP-2-carrying exosomes promoted miR-378a-3p level and inhibited osteoclast differentiation. MiR-378a-3p mimics inhibited the NF-κB/MAPK signaling, and the direct binding specificity between miR-378a-3p and TRAF6 was validated via dual-luciferase reporter assay. Mechanistically, GLP-2-carrying exosomes enhanced bone parameters, suppressed inflammation and NF-κB/MAPK signaling pathway, which were reversed by miR-378a-3p antagonist. CONCLUSION: GLP-2-carrying exosomes showed promise in treating osteoporosis by inhibiting osteoclast differentiation and modulating NF-κB/MAPK pathways via miR-378a-3p regulation. This novel axis presents a promising dual-targeted strategy for osteoporosis treatment, with engineered exosomes offering significant translational potential for bone regenerative therapy.